title: Hypersusceptibility mechanism of Tenofovir-resistant HIV to EFdA.
abstract: BACKGROUND
  The K65R substitution in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is the major resistance mutation selected in patients treated with first-line antiretroviral tenofovir disoproxil fumarate (TDF). 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), is the most potent nucleoside analog RT inhibitor (NRTI) that unlike all approved NRTIs retains a 3'-hydroxyl group and has remarkable potency against wild-type (WT) and drug-resistant HIVs. EFdA acts primarily as a chain terminator by blocking translocation following its incorporation into the nascent DNA chain. EFdA is in preclinical development and its effect on clinically relevant drug resistant HIV strains is critically important for the design of optimal regimens prior to initiation of clinical trials.
  
  
  RESULTS
  Here we report that the K65R RT mutation causes hypersusceptibility to EFdA. Specifically, in single replication cycle experiments we found that EFdA blocks WT HIV ten times more efficiently than TDF. Under the same conditions K65R HIV was inhibited over 70 times more efficiently by EFdA than TDF. We determined the molecular mechanism of this hypersensitivity using enzymatic studies with WT and K65R RT. This substitution causes minor changes in the efficiency of EFdA incorporation with respect to the natural dATP substrate and also in the efficiency of RT translocation following incorporation of the inhibitor into the nascent DNA. However, a significant decrease in the excision efficiency of EFdA-MP from the 3' primer terminus appears to be the primary cause of increased susceptibility to the inhibitor. Notably, the effects of the mutation are DNA-sequence dependent.
  
  
  CONCLUSIONS
  We have elucidated the mechanism of K65R HIV hypersusceptibility to EFdA. Our findings highlight the potential of EFdA to improve combination strategies against TDF-resistant HIV-1 strains.
authors.affiliation: Christopher Bond Life Sciences Center, Department of Molecular Microbiology & Immunology, University of Missouri, Columbia, MO 65211, USA.
authors.forename: Eleftherios
authors.initials: E
authors.lastname: Michailidis
authors.forename: Emily M
authors.initials: EM
authors.lastname: Ryan
authors.forename: Atsuko
authors.initials: A
authors.lastname: Hachiya
authors.forename: Karen A
authors.initials: KA
authors.lastname: Kirby
authors.forename: Bruno
authors.initials: B
authors.lastname: Marchand
authors.forename: Maxwell D
authors.initials: MD
authors.lastname: Leslie
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Huber
authors.forename: Yee T
authors.initials: YT
authors.lastname: Ong
authors.forename: Jacob C
authors.initials: JC
authors.lastname: Jackson
authors.forename: Kamalendra
authors.initials: K
authors.lastname: Singh
authors.forename: Eiichi N
authors.initials: EN
authors.lastname: Kodama
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Mitsuya
authors.forename: Michael A
authors.initials: MA
authors.lastname: Parniak
authors.forename: Stefan G
authors.initials: SG
authors.lastname: Sarafianos
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019380:Anti-HIV Agents / Q000494:pharmacology*; D002460:Cell Line; D003839:Deoxyadenosines / Q000494:pharmacology*; D024882:Drug Resistance, Viral; D054303:HIV Reverse Transcriptase / Q000037:antagonists & inhibitors / Q000235:genetics; D015497:HIV-1 / Q000187:drug effects*; D006801:Humans; D008826:Microbial Sensitivity Tests; D050505:Mutant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D020125:Mutation, Missense; D063065:Organophosphonates / Q000494:pharmacology*; D000068698:Tenofovir
chemical_list: D019380:Anti-HIV Agents; D003839:Deoxyadenosines; D050505:Mutant Proteins; D063065:Organophosphonates; D000068698:Tenofovir; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D000225:Adenine; C558823:islatravir